VACCINE STUDIES: A crucial element in the development of effective prophylactic strategies for AIDS is an experimental animal model in which the course of immunodeficiency virus infection parallels the pathogenesis of the human disease. SIV infection of macaques is a relevant model since it induces an immunodeficiency syndrome in infected macaques that is remarkably similar to human AIDS. Recent studies suggest that SIV-infection is likely to be more representative of human AIDS pathogenesis than SHIV viruses that utilize CXCR4 as their coreceptor and target naive rather than memory CD4+ T cells. During primary viremia in SHIV-infected macaques, massive elimination of CXCR4 naive CD4+ T cells occurred. In contrast, CCR5+ memory CD4+ T cells were selectively depleted in rapidly progressing SIV-infected macaques. Thus SHIV and SIV target different subsets of CD4+ T cells. These differences explain the different pathogenesis of SIV and SHIV. Importantly, in the context of developing an effective vaccine, regimens that suppress SHIV might not protect monkeys against SIV or humans against HIV. The early major vaccine effort within my laboratory has been the evaluation of the highly attenuated vaccinia virus Ankara (MVA) strain as a recombinant vector. We used MVA-SIV recombinants expressing Env, Gag-pol or both in three separate challenge studies in rhesus macaques. Macaques expressing the MamuA*01 MHC-Class I allele were used to evaluate cellular immune responses. Immunization with MVA-SIV recombinants resulted in a reduction in setpoint plasma viral load that was associated with prolonged survival. These data demonstrate that vaccination with MVA-SIV recombinants results in significant protection from high viremia and AIDS. These animals have been the focus of follow-up studies to determine the long-term efficacy of such vaccines. All but two vaccinees have progressed to AIDS by seven years after challenge suggesting that immune escape from such vaccines is a major concern. Recent studies have evaluated the effect of vaccination of memory CD4+ T cells in the blood; vaccination was associated with better preservation of memory CD4+ T cells and inversely correlated with virus load. Importantly, preservation of CD4+ T cells early post challenge correlated with improved survival and with titers of neutralizing antibodies to the challenge virus prior to challenge. All of our previous vaccine studies have used SIVsmH4 as an immunogen, which possesses a lab-adapted neutralization-sensitive envelope. Therefore, these previous studies have focused mostly on generating cellular immune responses. The purpose of future experiments will be to use a prime-boost strategy to generate antibody that will neutralize the pathogenic SIVsmE543 and study the role of this antibody in the SIV model. Prior studies have only demonstrated a role for neutralizing antibody in vivo using the less pathogenic SIVmne model or in SHIV. Similar studies using SIVmac239 have been hampered due to the neutralization resistance associated with its envelope glycoprotein. We therefore generated a MVA recombinant that expresses the less neutralization-resistant SIVsmE543-3 envelope and a plasmid of the codon-optimized gp140 that has been used to generate recombinant trimeric SIVsmE543-3 envelope. We are also evaluating neutralizing activity of a large IgG pool that was purified from a group of six SIVsmE660-infected macaques;this IgG effectively neutralizes SIVsmE660 and SIVsmH4 but does not cross-neutralize SIVsmE543-3. We plan to use this IgG in passive prophylactic experiments to determine the predictive power of in vitro neutralization assays. A non-integrating mutant, SIVsmD116N clone, was derived from SIVsmE543-3 by introducing an Asp (D) to Asn (N) mutation into the invariant D-116 integrase residue in the catalytic domain. This point mutation completely abolishes viral DNA integration of HIV without affecting other known viral functions such as reverse transcription and nuclear targeting. The SIVsmD116N and wild type SIVsmE543-3 clones were transfected into 293T cells to generate cell free virus and their replication was assessed in CEMx174 cells, macaque PBMC and macaque monocyte-derived macrophages (MDM). While the wild type virus replicated in each of these cell types, no RT activity was observed in cell-free media following infection with the integrase mutant. Alu-PCR confirmed that SIVsmD116N did not integrate into genomic DNA. The SIVsmD116N mutant was able to synthesize viral DNA with almost equal efficiency to wild type virus and viral DNA persisted in macrophages for as long as 30 days. The capacity of a nonintegrating SIV to persistently generate viral products suggests that nonintegrating lentiviral vectors could serve to express protein for vaccine purposes, without the permanency of an integrated retrovirus or disruption of normal cellular genes.